Cross-linking of estrogen receptor to chromatin in intact MCF-7 human breast cancer cells: optimization and effect of ligand.
To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.
Wrenn
C K
CK
Department of Physiology and Biophysics, University of Illinois, Urbana.
Katzenellenbogen
B S
BS
D001943:Breast Neoplasms / Q000235:genetics*; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives; D004965:Estrogen Antagonists / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D053284:Polyunsaturated Alkamides; D012319:RNA Polymerase II / Q000235:genetics; D011960:Receptors, Estrogen / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D012261:Ribonucleoproteins / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D002843:Chromatin; D003432:Cross-Linking Reagents; D004965:Estrogen Antagonists; D008024:Ligands; D053284:Polyunsaturated Alkamides; D011960:Receptors, Estrogen; D012261:Ribonucleoproteins; D005557:Formaldehyde; D004958:Estradiol; C051781:ICI 164384; D004247:DNA; D012319:RNA Polymerase II